Ion transport across the plasma membrane is critical for maintaining the normal physiology of the cell. Ion transport across the plasma membrane is mediated by a variety of membrane bound proteins which act as channels and pumps. Dysfunctional ion channels or pumps will lead to a disease state. Cystic fibrosis is a disease resulting from a defect in a cAMP-mediated chloride channel, CFTR (Welsh et al. (1993) Cell 73:1251-1254). The physiological manifestation of cystic fibrosis includes airway obstruction resulting from thick secretions of mucus into the airways of the lung and gastrointestinal tract and the subsequent colonization of the lung airways by pathogenic microorganisms (Clarke et al. (1992) Science 257:1125-1128; Clarke et al. (1994) Proc Natl Acad Sci USA 91:479-483; Eng et al. (1996) Ped Pulmonol 21:77-83) and mucus plugging of pancreatic ducts of the gastrointestinal tract (WO 01/54685).
Mucus is a thin film of protective viscoelastic liquid which lines the airways, gastrointestinal tract, and other organs containing mucus membranes. Mucus is an aqueous solution in which the major component is a glycoconjugate, known as mucin. Mucin secretion may be constitutive, regulated, or in response to external stimuli, in particular irritants.
The association of cystic fibrosis with aberrant ion transport has led investigators to hypothesize that dysfunctional ion transport might be related to other diseases with similar symptoms. Thus, dysfunctional ion transport has been implicated in diseases such as asthma and chronic obstructive pathway disease (COPD), i.e., emphysema and chronic bronchitis.
The Calcium-activated Chloride Channels (CaCC), also known as Chloride Channels, Calcium-Activated (CLCA) proteins, are emerging as a new class of channel proteins that mediate Ca2+-activated Cl− conductance in a variety of tissues. It has been reported that the stimulation of chloride secretion results in the secretion of mucin from goblet cells in the intestinal epithelium. (Halm, et al. (1995) Am. J. Physiol. 269:929-942.) Members of the CLCA family have been cloned, isolated, and partially characterized from human, bovine, and murine species. These proteins demonstrate a high degree of homology in their size, sequence, and predicted structure yet can vary considerably in tissue distribution.
Members of this family include: bovine lung endothelial cell adhesion molecule, Lu-ECAM-1 (Elble, et al. (1997) J. Biol. Chem. 272:27853-27861); bovine Ca2+-activated Cl−, CaCC or bCLCA1 (Cunningham, et al. (1995) J. Biol. Chem. 270:31016-31026); murine CLCA1, mCLCA1 (Gandhi, et al. (1998) J. Biol. Chem. 273:32096-32101); human CLCA1, hCLCA1 (Gruber, et al. (1998) Genomics 54:200-214); murine Gob-5, mGob-5, also known as murine CLCA3, mCLCA3 (Komiya, et al. (1999) Biochem. Biophys. Res. Comm. 255:347-351, Zhou et al. (2002) Am J. Respir. Cell Mol. Biol. 25:486-491); and human CLCA2, hCLCA2 (Gruber, et al. (1999) Am. J. Physiol. 276(Cell Physiol. 45):C1261-C1270.) Recently, Holroyd, et al., PCT publication No. WO 99/44620, described a calcium activated chloride channel that is induced by IL-9.
mCLCA3 is a 913 amino acid, approximately 110 kDa, glycosylated protein which is processed to an approximately 90 kDa amino terminal cleavage product and additional smaller fragments. mCLCA3 has been shown, through in situ hybridization, to be expressed in the mucus-secreting cells of the stomach, small intestine, colon, and uterus, along with slight expression in the trachea. (Komiya, et al. supra.)
The three human CLCA homologs (hCLCA1, hCLCA2, and hCLCA3) thus far cloned, isolated, and partially characterized, all retain sequence homology, similar cDNA length, and are all located on the short arm of chromosome 1 (1p22-p31). Human CLCA proteins show a restricted pattern of expression in differing secretory tissues. Human CLCA1 was the first reported calcium activated chloride channel in humans. The 31,902-bp hCLCA1 gene is located on chromosome 1p22-p31, contains 14 introns, and is preceded by a canonic promoter region that contains an LI transposable element. Expression of hCLCA1 is predominant in intestinal basal crypt epithelia and goblet cells. Expression has been detected in small intestine, colon, appendix, and rectal mucosa. (Ritzka et al. (2004) Hum. Genet. 115:483-491)
A protein processing model has been proposed for hCLCA1 in which the primary translation product (125-kDa) is cleaved to a 90-kDa and a group of 37- to 41-kDa proteins, the latter apparently representing different glycosylation products of the same polypeptide (Gruber et al., supra). Transient expression of hCLCA1 cDNA in HEK 293 cells is associated with an increase in whole-cell Ca2+-activated Cl conductance that is susceptible to inhibition with anion channel blocking compounds. Cell attached patch recordings of transfected cells in this study revealed single channels with a slope conductance of 13.4 pS (Gruber et al., supra). It has further been suggested that expression of hCLCA1 is associated with chloride ion flux across the plasma membrane and that non-selective chloride channel inhibitors such as niflumic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, and dithiothreitol will abrogate this effect. (Gruber et al., supra).
hCLCA1, and its proposed murine homolog, mCLCA3, are putative calcium activated chloride channels (WO 99/44620). Both have been implicated in the pathology associated with asthma. Asthma is characterized by a hypersensitivity to environmental allergens which is associated with an inflammatory response and the increased production of mucin (WO 01/54685; WO 99/44620). Expression of hCLCA1 and mCLCA3 is up-regulated in response to allergen challenge. Expression has also been linked to mucin overproduction (Hoshino et al. (2002) Am J Respir Crit. Care Med 165:1132-1136). Over expression of hCLCA1 and mCLCA3 has been shown to induce expression of MUC5AC, a mucin gene, in a muco-epidermal cell line. Expression of hCLCA1 was shown to be upregulated in patients with bronchial asthma compared with control subjects (Hoshino et al., supra; Toda et al. (2002) J. Allergy Clin. Immunol. 109:246-250). Additionally, adenovirus mediated antisense therapy has abrogated the effects of mCLCA3 hyper-responsiveness and mucin production in an in vivo mouse model (Nakanishi et al. (2001) Proc Natl Acad Sci USA 98:5175-5180).
Hegab et al. (2004; J. Med. Genet. 41:e27) have suggested a role for hCLCA1 in chronic obstructive pulmonary disease. Both hCLCA1 and mCLCA3 are proposed to have roles in regulating antigen-stimulated epithelial cell functions in allergen-induced disease (Zhou et al., supra).
PDZ domains are regions of signaling proteins that function to modulate protein-protein interactions such as protein-protein recognition. Proteins containing PDZ domains have roles in many cell functions, including cell signaling, cell adhesion, ion transport, and formation of tight junctions. Structurally, PDZ domains are 80-90 amino acid modular domains that comprise six beta-strands (betaA to betaF) and two alpha-helices, A and B, compactly arranged in a globular structure. Peptide binding of the ligand takes place in an elongated surface groove as an anti-parallel beta-strand interacts with the betaB strand and the B helix. The structure of PDZ domains allows binding to a free carboxylate group at the end of a peptide through a carboxylate-binding loop between the betaA and betaB strands. PDZ domains bind to the last four to six carboxy-terminal amino acids in a target protein, with different classes of PDZ proteins recognizing different characteristic sequence motifs. Some PDZ domains can also recognize internal motifs present in beta-hairpin structures. (Ponting, C. P. et al. (1997) Bioessays 19:469-479; Doyle et al. (1996) Cell 85:1067-1076; Songyang, Z. et al. (1997) Science 275:73-77; Bezprozvanny, I. and Maximov, A. (2001) FEBS Lett. 509:457-462; and Jelen, F. et al. (2003) Acta Biochimica Polonica 50:985-1017).
The majority of PDZ proteins are associated with the clustering and localization of proteins at the plasma membrane. Interestingly, PDZ proteins have been shown to be involved in the control of sorting, localization, and release from the secretory pathway (Scott, D. B. et al. (2001) J. Neurosci. 21:3063-3072; Greger, I. H. (2002) Neuron 34:75-772).
Modulators of interactions between PDZ proteins and CLCA1 proteins are useful in the treatment of pulmonary diseases including asthma, chronic obstructive pathway disease, including emphysema and chronic bronchitis, and disorders associated with improper mucus secretion. PDZ proteins are useful in the detection of CLCA1 proteins and in the diagnosis of pulmonary diseases including asthma, chronic obstructive pathway disease, including emphysema and chronic bronchitis, and disorders associated with improper mucus secretion.